As one form of a dry analysis element, numerous kinds of integral multilayer analysis elements (hereinafter often referred to as a "multilayer analysis element") have been proposed which are formed by laminating a water-absorbing coloring reagent layer comprising a coloring reagent dispersed in a hydrophilic polymer binder, on a transparent light-transmissive water-impermeable support, and laminating a porous spreading layer (hereinafter often referred to as a "spreading layer") adhered thereover as an outermost layer. The spreading layer of the multilayer analysis element is a layer having the function of spreading an aqueous liquid sample (for example, blood such as whole blood, plasma, and serum, lymphatic fluid, saliva, spinal fluid, vaginal fluid, urine, drinking water, alcoholic drinks, river water, various drainages, etc). The aqueous liquid sample spotted on the upper surface of the element (which is in the side remote from the transparent support) is spreaded to the surface direction without substantially localizing the components contained in the liquid sample so that the thus spreaded liquid sample may be transferred into the water-absorbing hydrophilic polymer binder and layer, which is typically a water-absorbing coloring reagent layer, almost at a constant ratio per unit surface area. This function of the spreading layer is referred to as a metering function.
Japanese Patent Application (OPI) No. 6167/87 (the term "OPI" as used herein means a "published unexamined Japanese patent application") describes a integral multilayer analysis element for determination of total lactic dehydrogenase, which contains a fluorinated surfactant. In the multilayer analysis element, a fluorinated surfactant is incorporated into one or more layers of the spreading layer, subbing layer, reagent layer and registration layer, whereby an improvement in accuracy (that is, a decrease of the deviation coefficient value caused by contingent error) could be attained. However, the porous spreading layer in the multilayer analysis element is limited only to a non-fibrous isotropic porous spreading layer, such as a fine continuous pore-containing non-fibrous isotropic porous spreading layer formed by polymer microbeads, glass microbeads or diatomaceous earth supported by a hydrophilic polymer binder (as described in Japanese Patent Publication No. 21677/78, U.S. Pat. No. 3,992,158, etc.) or a fine continuous pore-containing porous layer formed by adhering polymer microbeads in a spot-like manner with a polymer adhesive which does not swell with water (that is, a three-dimensional lattice-like granule-structural layer, as described in Japanese Patent Application (OPI) No. 90859/80).
It is known that the addition of a surfactant, preferably a nonionic surfactant such as p-nonyl phenoxy polyethoxy ethanol, etc., to the spreading layer is effective for improving the metering function of the layer with respect to the aqueous liquid sample to be tested. However, it is also known that the range of the metering function which can be improved by the addition of the nonionic surfactant is narrow.
On the other hand, the improvement of accuracy (that is, a decrease of the deviation coefficient value caused by contingent error) in the integral multilayer analysis element for determination of total lactic dehydrogenase (LDH) described in Japanese Patent Application (OPI) No. 6167/87, which contains fluorinated surfactant is caused by the association of the fluorinated surfactant with the reaction between the LDH, which is an analyte (that is, a component to be analyzed), and a substrate for LDH, in the system comprising the combination of the non-fibrous isotropic porous spreading layer and the fluorinated surfactant, whereby the analysis accuracy is improved within the range of a low LDH level.